1. Antibodies
  2. Homo sapiens
  3. CBFB

ENCAB725LFJ

Antibody against Homo sapiens CBFB

Homo sapiens
K562, GM12878, HepG2, MCF-7
characterized to standards with exemption
Status
released
Source (vendor)
GeneTex
Product ID
GTX106820
Lot ID
39981
Characterized targets
CBFB (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
affinity
Isotype
IgG
Antigen description
Recomb fragment corresp to a region within amino acids 1 and 182 of PEBP2beta (Uniprot ID#Q13951)
External resources

Characterizations

CBFB (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
Attachment from submitters
compliant
Caption
GM12878 whole cell lysate was immunoprecipitated using the primary antibody (GeneTex; GTX106820). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. The whole lane was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of whole lane from GM12878: The sample was analyzed on a LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectral analysis with probability based matching at p < 0.05. SEQUEST results were reported with ProteinProphet protXML Viewer (TPP v4.4 JETSTREAM) and filtered for a minimum probability of 0.9. All protein hits that met these criteria were reported, including common contaminants. Fold enrichment for each protein reported was determined using a custom script based on the FC-B score calculation from the reference Mellacheruvu et al., 2013. The CRAPome: a contaminant repository for affinity purification mass spectrometry data. Nat. Methods. 10(8):730-736. Doi:10.1038/nmeth.2557. The target protein, CBFB, was identified as the 8th enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitter comment
In some cases, we exercised the option of extracting the whole lane to send for IP-mass spectrometry as opposed to cutting and sending discrete bands from a gel. This is especially the case when the antibody results in multiple and non-discrete bands or no visible bands in the predicted size range of the protein when observed by IP-Western. To ensure that the targeted protein can be identified in the IP, whole cell lysates are loaded in a 12% Bio-Rad TGX gel and run for 5-10 minutes using the Bio-Rad Tetra Cell system. The entire gel lane or just the single unseparated band is excised from the gel and sent for analysis. We do not provide coomassie blue-stained gel images in these instances as all one observes is a thick, unresolved band near the loading well.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 CBFB-WL.png
CBFB (Homo sapiens)
K562GM12878HepG2MCF-7
Method: immunoblot
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Whole cell lysates of K562, GM12878, HepG2, and MCF7 were immunoprecipitated using the primary antibody (GeneTex; GTX106820). The IP fraction was separated on a 12% acrylamide gel with the Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. The membrane was probed with the primary antibody (same as that used for IP) and a secondary HRP-conjugated antibody. The resulting bands were visualized with SuperSignal West Femto Solution (Thermo Scientific). Protein Marker (PM) is labeled in kDa. A single prominent band was detected at ~22 kDa.
Submitter comment
In some cases, we exercised the option of extracting the whole lane to send for IP-mass spectrometry as opposed to cutting and sending discrete bands from a gel. This is especially the case when the antibody results in multiple and non-discrete bands or no visible bands in the predicted size range of the protein when observed by IP-Western. To ensure that the targeted protein can be identified in the IP, whole cell lysates are loaded in a 12% Bio-Rad TGX gel and run for 5-10 minutes using the Bio-Rad Tetra Cell system. The entire gel lane or just the single unseparated band is excised from the gel and sent for analysis. We do not provide coomassie blue-stained gel images in these instances as all one observes is a thick, unresolved band near the loading well.
Reviewer comment
Not within 20% of expected size. Rescued by mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 CBFB_IP-WB.png